ABSTRACT Constitutively increased intracellular pH (pHi) is common to most cancers regardless of their tissue origin or genetic background. However, how a higher pHi enables disease progression and the molecular mechanisms mediating pHi-dependent cancer cell behaviors are understudied and mostly not understood. We previously revealed how increased pHi can enable metastatic progression by regulating pH-sensing proteins controlling directed cell migration. We now will address how increased pHi enables three additional cancer cell behaviors: tumor formation, metabolic reprograming and retention of recurring somatic mutations. We exploit our unique expertise in resolving at the molecular, cellular and tissue levels how pHi dynamics regulates cell functions. We have a strong track record of bridging structural and cell biology to reveal the design principles and functional significance of pH sensors, defined as proteins with activities or ligand binding affinities regulated within the narrow pH range of the cell. To identify pH sensors we integrate analyses of signaling pathways, cancer mutations databases, and titrating networks of ionizable residues in proteins with molecular dynamics simulations, biochemistry and cell physiology. Our expertise in measuring real-time pHi dynamics in vivo using a genetically encoded biosensor brings a distinct new approach to understanding cancer cell biology. In Aim 1 we will test the hypothesis that increased pHi is necessary and sufficient for tumorigenic behaviors. We will resolve mechanisms for pHi-dependent tumorigenesis in mouse models to determine the synthetic lethality we reported in Drosophila with loss of H+ efflux and oncogene expression. We will determine how increased pHi in the absence of oncogenes induces dysplasia, and measure spatial and temporal pHi dynamics during tumorigenesis as a new metric to inform us about heterogeneity of tumor cells. These studies include a structural and functional analysis of pH sensors predicted to enable oncogenic behaviors. In Aim 2 we will test the hypothesis that increased pHi enables metabolic reprograming in cancer. We will determine mechanisms for how increased pHi can promote a switch in glucose utilization from mitochondrial oxidative phosphorylation to increased aerobic glycolysis. These studies include a structural and functional analysis of the recognized pH-sensitive glycolytic enzymes phosphofructokinase-1 and lactate dehydrogenase. We also resolve how increased pHi suppresses mitochondrial oxidative phosphorylation and regulates carbon fates, determined by magnetic resonance spectroscopy. In Aim 3 we will test the hypothesis that increased pHi provides a selective pressure for the retention of somatic mutations with histidine residues. These studies are supported by findings from an R21 award that verified pH sensitivity of recurring somatic mutations in cancers and with bioinformatics analyses identified cancer subtypes based on amino acid substitution signatures, which we will test for shared functional properties. Outcomes of our proposal will generate new insights on molecular mechanisms enabling cancer that can inform therapeutic approaches targeting pH sensors to limit disease progression.